Cell-free (9), cell-based, and animal-based (10) systems each have several advantages and disadvantages (4,5,11), but, despite the number of expression systems currently available, no native human GPCR has yet been crystallized. rhodopsin (Rho) obtained from bovine retina. Generally high expression levels of 11 native and mutant GPCRs demonstrated the potential of thisC. eleganssystem to produce milligram quantities of high-quality GPCRs and possibly other membrane proteins suitable for detailed characterization.Salom, D., Cao, P., Sun, W., Kramp, K., Jastrzebska, B., Jin, H., Feng, Z., Palczewski, K. Heterologous expression of functional G-protein-coupled receptors inCaenorhabditis elegans. Keywords:native GPCR, purification, membrane proteins Modern pharmaceutical research relies increasingly on structural information about protein targets. Both lead compound generation and optimization are greatly facilitated if a 3-dimensional structure of the biological target is known. Although membrane proteins (MPs) comprise the majority of druggable targets (1), they account for Amidopyrine <1% of structures in the Protein Data Base (PDB). Human G-protein-coupled receptors (GPCRs) in particular are targets for about half of therapeutic drugs currently sold or in development (15). Despite the biomedical significance of GPCRs, bovine rhodopsin [(b)Rho] is the only vertebrate GPCR with an unaltered native sequence whose structure has been solved to atomic resolution (68). Rhodopsin can be obtained in reasonable amounts from its natural source, whereas other vertebrate GPCRs are expressed at low levels in Rabbit polyclonal to RAB1A their native tissues. A number of expression systems have been developed that yield GPCRs in quantities sufficient for crystallization. Cell-free (9), cell-based, and animal-based (10) systems each have several advantages and disadvantages (4,5,11), but, despite the number of expression systems currently available, no native human GPCR has yet been crystallized. Common problems with eukaryotic expression systems are their low yield, high cost, and either lack or heterogeneity of post-translational modifications. Decades of research on GPCRs with diffusible ligands are starting to yield important structural information (12). Advances range from improvements in GPCR expression and purification, stabilizing mutations, and identification of new ligands and detergents, to new methods for GPCR crystallization and X-ray diffraction collection (1315). However, to date these improvements have been made at the expense of important changes Amidopyrine in the target protein. Such changes include chemical modifications, deglycosylation, multiple point mutations, long deletions, insertions of entire proteins,etc., producing GPCRs with altered pharmacological and functional properties compared to those of their natural human counterparts. High-resolution structures of native proteins remain the most reliable templates for precise structure-based drug design. Therefore, much effort is still needed to develop new strategies for the expression and purification of milligram amounts of native GPCRs in a homogeneous, functional form. Here we report a new expression system for heterologous human GPCRs inCaenorhabditis elegansthat is potentially applicable to other MPs. This nematode expresses 1100 GPCRs (5% of its genome; ref.16) in neurons to detect bacteria and environmental compounds. Heterologous expression of human GPCRs inC. eleganshas certain advantages over otherin vivoandin vitroexpression systems because of this animal’s relatively facile genetic manipulation, short life cycle, scalability, phenotypic diversity, and potential tissue-specific MP expression. Thus,C. eleganscombines the advantages ofin vivoanimal expression and conventional single-cell expression systems. The demonstrated expression of several native and engineered GPCRs inC. elegansand simple purification of (b)isoRho and (h)A2AR illustrate the potential of this organism to become a major expression system for functional and structural studies of eukaryotic MPs in general and human GPCRs in particular. == MATERIALS AND METHODS == == GPCR gene constructs == GPCR expression constructs were generated as follows: promotersmyo-3(17) orH20(18) were inserted into a pBluscript KS(+) vector atHindIII/Xbal or Pstl, respectively. Synthesized cDNAs (Genescript, Piscataway, NJ, USA), Amidopyrine either codon optimized (19) or unoptimized, encoding a GPCR followed by a tobacco etch virus (TEV) protease site, T7 and Rho9 tags (20), and the polyadenine [poly(A)] tail ofunc-54(21), were engineered betweenNotl andXhol. Several constructs with modifications were exceptions, based on the following reasons: the TEV cleavage site and T7 and Rho9 tags were not introduced into the (b)opsin construct because it contained a Rho9 tag at the end of its C terminus; previously crystallized constructs of mutated (m) GPCRs contained no TEV site, T7 nor Rho9, because they already had His tags; and the codon-unoptimized (h)A2AR construct contained just the Rho9 tag (seeTable 1). The entire GPCR fusion protein.